Mono-ADP-ribosylation is a reversible modification of proteins, with NAD: arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing opposing arms of an ADP-ribosylation cycle. Prior studies have examined the structure of the mouse ADP-ribosylarginine hydrolase gene. The 5 flanking promoter region was shown to have a critical regulatory element at -199 to -89. This region was found to function in a number of cell lines, suggesting the requirement for a relatively common transcription factor. Further mutational analysis of the promoter and electrophoretic mobility shift assays identified a positive GC-box element (-107- -95); using supershift assays, Sp1 and Sp3 were found to bind to this motif. In cotransferction experiments using Drosophila SL2 cells, which lack endogenous Sp1, Sp1 was found to transactivate the ADP-ribosylarginine hydrolase promoter in a manner dependent on the presence of an intact Sp1-binding motif. The promoter activity pattern and involvement of Sp1 transcription factors are consistent with prior observations showing widespread hydrolase expression in mammalian tissues. - ADP-ribosylation, ADP- ribosylarginine hydrolase